Our OBJECTIVE is to determine the molecular mechanisms by which fluid flow guides the formation and growth of lymphatic valves. Valves ensure one-way fluid flow in the lymphatic system, and are essential to physiological function. Relatively little is known about how lymphatic valves form. This knowledge gap, coupled with the limited regenerative ability of the lymphatic system, represents a central roadblock in the development of effective treatments for lymphedema, a debilitating condition for which there is no cure. Understanding the molecular basis of lymphatic valvulogenesis would thus have a transformative impact on the treatment of lymphedema and other diseases of the lymphatic system. RATIONALE: Lymphatic valves form preferentially near vessel junctions. These regions feature complex, recirculating flow that is not present in straight portions of the lymphatic vasculature. We hypothesized that these flow patterns might provide a critical cue that triggers valve formation specifically in these locations. To test this hypothesis, we developed a unique in vitro assay that exposes lymphatic endothelial cells (LECs) to spatial gradients in wall shear stress (WSS) that mimic those found at the sites of valve formation. Remarkably, LECs exposed to this flow pattern recapitulate the migratory, morphogenetic, and signaling events that occur during the initial stages of valve formation in vivo. Further, we find that these responses depend on activation of sphingosine-1-phosphate receptor 1 (S1PR1) a GPCR that is activated by fluid flow in blood endothelial cells, and that is known to play an important role in lymphangiogenesis. These and other preliminary data strongly suggest that spatial patterns in WSS play a central role in sculpting lymphatic valve development. STRATEGY: We have created and characterized in vitro culture systems that expose LECs to key attributes of the flow environment found at sites of valve formation. These devices provide quantitative control of the flow stimuli experienced by the LECs, allow time-lapse, multi-day imaging, and provide high experimental throughput, capabilities that are difficult to attain in an in vivo setting. This combination of attributes is nique in its ability to uncover the molecular mechanisms by which LECs sense and respond to fluid flow. The GOALS of our research are: Aim 1. Elucidate the role of fluid flow in guiding lymphatic valve formation. We will determine the role of WSS gradients and oscillating flow in shaping lymphatic valve development (Aim 1a), and discover how signaling pathways known to be required for valvulogenesis are coupled to LEC flow sensing (Aim 1b). Further, we will create 3D cell culture systems that fully recapitulate the flow environment found at sites of valve growth, and use this powerful technology to recreate key attributes of valvulogenesis in vitro. Aim 2. Determine the molecular mechanism by which S1PR1 mediates flow sensing in LECs. We will elucidate the role of flow-activated S1PR1 signaling in valve formation (Aim 2a), and use cell biological and biophysical approaches to determine the molecular mechanism by which flow activates S1PR1 (Aims 2b and 2c).